The nucleotide sequence of recG, the distal spo operon gene in Escherichia coli K-12.

A gene is identified in the Escherichia coli K-12 spo operon as recG. Previously identified genes in the spo operon were spoS, alias rpoZ, encoding the omega (omega) subunit of RNA polymerase, as well as the spoT gene encoding the major cellular source of guanosine 3',5'-bispyrophosphate hydrolase activity. The gene order within the spo operon is: spoS (rpoZ), spoT, spoU, recG. A convergent gltS gene is present beyond the spo operon. Mutants bearing recG deletion-insertion alleles display mild sensitivities to both ultraviolet irradiation and to mitomycin C, which is expected to be due to a known recG insertion allele. Deletion-insertion mutations in upstream operon genes (spoT and spoU) show polar effects on these assays of recG function. The deduced 693-amino acid (aa) RecG sequence shows a weak, but significant, relatedness to aa sequence motifs previously reported for putative helicases involved in replication, recombination, and DNA repair.     

A mutation in helicase motif III of E. coli RecG protein abolishes branch migration of Holliday junctions.

The RecG protein of Escherichia coli catalyses branch migration of Holliday junctions made by RecA and dissociates synthetic X junctions into duplex products in reactions that require hydrolysis of ATP. To investigate the mode of action of this enzyme a chromosomal mutation that inactivates recG (recG162) was cloned and sequenced. The recG162 mutation is a G:C to A:T transition, which produces an Ala428 to Val substitution in the protein. This change affects a motif (motif III) in the protein that is highly conserved in DNA and RNA helicases. RecG162 protein was purified and shown to retain the ability to bind synthetic X and Y junctions. However, it does not dissociate these junctions and fails to catalyse branch migration of Holliday junction intermediates purified from a RecA strand exchange reaction. RecG162 retains a DNA-dependent ATPase activity, but this is much reduced relative to the wild-type protein, especially with single-stranded DNA as a co-factor. These results suggest that branch migration by RecG is related to a junction-targeted DNA helicase activity.     

Effect of host species on recG phenotypes in Helicobacter pylori and Escherichia coli

Recombination is a fundamental mechanism for the generation of genetic variation. Helicobacter pylori strains have different frequencies of intragenomic recombination, arising from deletions and duplications between DNA repeat sequences, as well as intergenomic recombination, facilitated by their natural competence. We identified a gene, hp1523, that influences recombination frequencies in this highly diverse bacterium and demonstrate its importance in maintaining genomic integrity by limiting recombination events. HP1523 shows homology to RecG, an ATP-dependent helicase that in Escherichia coli allows repair of damaged replication forks to proceed without recourse to potentially mutagenic recombination. Cross-species studies done show that hp1523 can complement E. coli recG mutants in trans to the same extent as E. coli recG can, indicating that hp1523 has recG function. The E. coli recG gene only partially complements the hp1523 mutation in H. pylori. Unlike other recG homologs, hp1523 is not involved in DNA repair in H. pylori, although it has the ability to repair DNA when expressed in E. coli. Therefore, host context appears critical in defining the function of recG. The fact that in E. coli recG phenotypes are not constant in other species indicates the diverse roles for conserved recombination genes in prokaryotic evolution.     

A Corynebacterium glutamicum rnhA recG double mutant showing lysozyme-sensitivity, temperature-sensitive growth, and UV-sensitivity

Corynebacterium glutamicum mutant KY9707 was originally isolated for lysozyme-sensitivity, and showed temperature-sensitive growth. Two DNA fragments from a wild-type C. glutamicum chromosomal library suppressed the temperature-sensitivity of KY9707. These clones also rescued the lysozyme-sensitivity of KY9707, although partially. One of them encodes a protein of 382 amino acid residues, the N-terminal domain of which was homologous to RNase HI. This gene suppressed the temperature-sensitive growth of an Escherichia coli rnhA rnhB double mutant. We concluded that this gene encodes a functional RNase HI of C. glutamicum and designated it as rnhA. The other gene encodes a protein of 707 amino acid residues highly homologous to RecG protein. The C. glutamicum recG gene complemented the UV-sensitivity of E. coli recG258::kan mutant. KY9707 showed increased UV-sensitivity, which was partially rescued by either the recG or rnhA gene of C. gluamicum. Point mutations were found in both recG and rnhA genes in KY9707. These suggest that temperature-sensitive growth, UV-sensitivity, and probably lysozyme-sensitivity also, of KY9707 were caused by mutations in the genes encoding RNase HI and RecG.     

radC102 of Escherichia coli is an allele of recG

The radC102 mutation causes mild UV and X-ray sensitivity and was mapped previously to near pyrE and recG at 82 min on the Escherichia coli chromosome (I. Felzenszwalb, N. J. Sargentini, and K. C. Smith, Radiat. Res. 97:615-625, 1984). We report that radC102 has two striking phenotypes characteristic of recG mutations. First, it causes dramatically increased RecA-dependent mutation in a stationary-phase mutation assay. Second, it causes extreme UV sensitivity in combination with ruv mutations affecting the RuvABC Holliday junction resolution system. DNA sequencing of the radC and recG genes in radC102 strains revealed that the radC102 mutation creates a stop codon in recG that is predicted to truncate the RecG protein at 410 of 603 amino acids. A low-copy-number plasmid carrying the radC(+) gene did not affect the UV sensitivity of a wild-type strain, a radC102 strain, or a recG258::Tn10mini-kan strain. We conclude that radC102 is an allele of recG and that the function of the RadC protein remains to be determined.     

Branch migration of Holliday junctions: identification of RecG protein as a junction specific DNA helicase.

The product of the recG gene of Escherichia coli is needed for normal recombination and DNA repair in E. coli and has been shown to help process Holliday junction intermediates to mature products by catalysing branch migration. The 76 kDa RecG protein contains sequence motifs conserved in the DExH family of helicases, suggesting that it promotes branch migration by unwinding DNA. We show that RecG does not unwind blunt ended duplex DNA or forked duplexes with short unpaired single-strand ends. It also fails to unwind a partial duplex (52 bp) classical helicase substrate containing a short oligonucleotide annealed to circular single-stranded DNA. However, unwinding activity is detected when the duplex region is reduced to 26 bp or less, although this requires high levels of protein. The unwinding proceeds with a clear 3' to 5' polarity with respect to the single strand bound by RecG. Substantially higher levels of unwinding are observed with substrates containing a three-way duplex branch. This is attributed to RecG's particular affinity for junction DNA which we demonstrate would be heightened by single-stranded DNA binding protein in vivo. Reaction requirements for unwinding are the same as for branch migration of Holliday junctions, with a strict dependence on hydrolysis of ATP. These results define RecG as a new class of helicase that has evolved to catalyse the branch migration of Holliday junctions.     

Conjugational recombination in resolvase-deficient ruvC mutants of Escherichia coli K-12 depends on recG.

ruvC mutants of Escherichia coli appear to lack an activity that resolves Holliday intermediates into recombinant products. Yet, these strains produce close to normal numbers of recombinants in genetic crosses. This recombination proficiency was found to be a function of recG. A "mini-kan" insertion in recG was introduced into ruvA, ruvB, and ruvC strains. Conjugational recombination was reduced by more than 100-fold in recG ruvA::Tn10, recG ruvB, and recG ruvC strains and by about 30-fold in a recG ruvA strain carrying a ruvA mutation that is not polar on ruvB. The double mutants also proved very deficient in P1 transduction and are much more sensitive to UV light than ruv single mutants. Since mutation of recG alone has very modest effects on recombination and sensitivity to UV, it is concluded that there is a functional overlap between the RecG and Ruv proteins. However, this overlap does not extend to circular plasmid recombination. The possibility that RecG provides a second resolvase that can substitute for Ruv is discussed in light of these findings.     

Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression.

The spoT gene of Salmonella typhimurium has been identified. Mutations in spoT map between gltC and pyrE at 79 min. The spoT1 mutant has elevated levels of guanosine 5'-diphosphate-3'-diphosphate (ppGpp) during steady-state growth and exhibits a slower than normal decay of ppGpp after reversal of amino acid starvation. The spoT1 mutation elevates his operon expression but is distinct from known his regulatory mutations. Elevated his operon expression in spoT mutants causes resistance to the histidine analogs, 1,2,4-triazole-3-alanine and 3-amino-1,2,4-triazole. These properties of spoT mutants allowed us to identify and characterize additional spoT mutants. Approximately 40% of these mutants are temperature sensitive for growth on minimal medium, suggesting that the spoT function is essential or that excessive accumulation of ppGpp is lethal.     

Bacillus subtilis RecG branch migration translocase is required for DNA repair and chromosomal segregation

The absence of Bacillus subtilis RecG branch migration translocase causes a defect in cell proliferation, renders cells very sensitive to DNA-damaging agents and increases approximately 150-fold the amount of non-partitioned chromosomes. Inactivation of recF, addA, recH, recV or recU increases both the sensitivity to DNA-damaging agents and the chromosomal segregation defect of recG mutants. Deletion of recS or recN gene partially suppresses cell proliferation, DNA repair and segregation defects of DeltarecG cells, whereas deletion of recA only partially suppresses the segregation defect of DeltarecG cells. Deletion of recG and ripX render cells with very poor viability, extremely sensitive to DNA-damaging agents, and with a drastic segregation defect. After exposure to mitomycin C recG or ripX cells show a drastic defect in chromosome partitioning (approximately 40% of the cells), and this defect is even larger (approximately 60% of the cells) in recG ripX cells. Taken together, these data indicate that: (i) RecG defines a new epistatic group (eta), (ii) RecG is required for proper chromosomal segregation even in the presence of other proteins that process and resolve Holliday junctions, and (iii) different avenues could process Holliday junctions.     

Modulation of recombination and DNA repair by the RecG and PriA helicases of Escherichia coli K-12.

The RecG protein of Escherichia coli is a structure-specific DNA helicase that targets strand exchange intermediates in genetic recombination and drives their branch migration along the DNA. Strains carrying null mutations in recG show reduced recombination and DNA repair. Suppressors of this phenotype, called srgA, were located close to metB and shown to be alleles of priA. Suppression depends on the RecA, RecBCD, RecF, RuvAB, and RuvC recombination proteins. Nine srgA mutations were sequenced and shown to specify mutant PriA proteins with single amino acid substitutions located in or close to one of the conserved helicase motifs. The mutant proteins retain the ability to catalyze primosome assembly, as judged by the viability of recG srgA and srgA strains and their ability to support replication of plasmids based on the ColE1 replicon. Multicopy priA+ plasmids increase substantially the recombination- and repair-deficient phenotype of recG strains and confer similar phenotypes on recG srgA double mutants but not on ruvAB or wild-type strains. The multicopy effect is eliminated by K230R, C446G, and C477G substitutions in PriA. It is concluded that the 3'-5' DNA helicase/translocase activity of PriA inhibits recombination and that this effect is normally countered by RecG.     

Error-prone DNA polymerase IV is regulated by the heat shock chaperone GroE in Escherichia coli

An insertion in the promoter of the operon that encodes the molecular chaperone GroE was isolated as an antimutator for stationary-phase or adaptive mutation. The groE operon consists of two genes, groES and groEL; point mutations in either gene conferred the same phenotype, reducing Lac+ adaptive mutation 10- to 20-fold. groE mutant strains had 1/10 the amount of error-prone DNA polymerase IV (Pol IV). In recG+ strains, the reduction in Pol IV was sufficient to account for their low rate of adaptive mutation, but in recG mutant strains, a deficiency of GroE had some additional effect on adaptive mutation. Pol IV is induced as part of the SOS response, but the effect of GroE on Pol IV was independent of LexA. We were unable to show that GroE interacts directly with Pol IV, suggesting that GroE may act indirectly. Together with previous results, these findings indicate that Pol IV is a component of several cellular stress responses.     

Involvement of DNA helicases in chromate resistance by Pseudomonas aeruginosa PAO1

Chromate-hypersensitive mutants of the Pseudomonas aeruginosa PAO1 strain were isolated using transposon insertion mutagenesis. Comparison of the nucleotide sequences of the regions interrupted within the PAO1 genome showed that mutant strains GGP-64 and AJ-22 were affected in open reading frames PA0967 and PA5345, which correspond to the ruvB and recG genes, respectively. These genes encode helicases RuvB and RecG involved in DNA replication, recombination and repair. The chromate resistance phenotype in mutants GGP-64 and AJ-22 was restored by cosmids bearing wild type ruvB or recG genes, respectively. Also, both mutant strains showed an increased susceptibility to the toxic oxyanions tellurite and selenite as well as to mitomycin C, but not to arsenite, paraquat and hydrogen peroxide. It was concluded that P. aeruginosa RuvB and RecG helicases are involved in repairing DNA damage caused by chromate or its derivatives.     

Cloning, sequencing, and characterization of the iturin A operon

Bacillus subtilis RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC. The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene, ituB, and the fourth gene, ituC, encode 609- and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by B. subtilis ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are D-Ser-->L-Asn, while in iturin A these amino acids are inverted (i.e., D-Asn-->L-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that ituD, ituA, and ituB have high levels of homology to the counterpart genes fenF (79%), mycA (79%), and mycB (79%), respectively. Although the overall level of homology of the amino acid sequences encoded by ituC and mycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B. subtilis 168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred. When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.